The overall effort of the laboratory-based research is to provide mechanistic interpretation of the findings obtained from the clinical protocols developed and implements by our group. Specifically, the underlying research goal is the characterization of the role of thyroid hormone action and its local, tissue-specific modulation in the delivery of time- and tissue specificity of the signal. Our laboratory is currently characterizing in vitro and ex vivo the functional activity of common polymorphisms of type-2 deiodinase gene. In order to achieve this goal, we have established a cell culture-based type-2 deiodinase expression system. Standard biochemistry methods are utilized for the enzymatic activity assay; protein/protein and nucleic acids/protein interactions are tested by immunoprecipitation and mobility shift assay. A common polymorphism in the 5UTR region of the DIO2 gene (258 A/G DIO2) has been associated with a shift in the ratio of circulating T3/T4, suggesting an increased in the activity of the enzyme in vivo leading to a shift of the reaction equilibrium. Our in vitro and ex vivo data, consistent with others genotype/phenotype association studies, indicate that the 258 A/G DIO2 variant induces an increase in the transcription of the gene by displacing a putative repressor. Current efforts are aimed to characterize the putative repressor factor interacting with the polymorphism. Type-2 deiodinase assay in primary culture of follicular thyroid cells. Collaborative work carried out with Dr. Gershengorns group (NIDDK-CEB) has led to the development of a reliable assay for the measurement of type-2 deiodinase activity in primary cultures of thyroid cells as a read-out of TSH-cAMP pathway. This system has been successfully utilized to test the activity of agonists of the TSH receptor. Development of a cell-based system to characterize the effects of substances modulating the type-2 deiodinase activity. We are currently developing cell line stably expressing type-2 deiodinase and a Thyroid Hormone Responsive Element (TRE)-driven reporter construct in order to evaluate the activity of small substances modulating the type-2 deiodinase activity. Pre-adipocyte differentiation and culture. During the past three FYs we have focused our effort in developing a system of re-differentiation of human adipocytes from stromal cells obtained during adipose tissue biopsy. This system will ultimately serve as a platform to test in a controlled fashion the effects of specific genotypes on adipose tissue function independently of the metabolic status of the subject at the time of the sampling. During FY13 we have successfully used this experimental model to characterize the role of hormones and adipokines in the differentiation of the adipocytes and in the modulation of their transcriptosome and functional activity. Further effort is directed toward characterizing the transcriptional pattern of this system throughout the differentiation process with a particular focus on the expression of brown-fat specific genes. We are now characterizing the differential molecular signature of pre-adipocytes harvested from different anatomical locations, as well as their potential for differentiation in brown adypocytes. Recent data published from our laboratory indicate that FGF-12, a brown adipose tissue-specific adipokine is able to promote the differentiation program toward a brown phenotype. The data have also been confirmed by functional assays including direct measurement of thermal activity.